Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

Podsumowanie

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

Streszczenie

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Wprowadzenie

The overall goal of this approach is to enable researchers to quickly measure the electrogenesis of the central nervous system (CNS) in the model insect, Drosophila melanogaster. This method is reliable, quick, and cost-efficient to perform physiological and toxicological experimentation. The CNS is essential for life and therefore, the physiological pathways critical for proper neural function have been explored extensively in an effort to understand or modify neural function. Characterization of the signaling pathways within the arthropod CNS has enabled the discovery of several chemical classes of insecticides that disrupt invertebrate neural function to induce mortality while limiting off-target consequences. Thus, the ability to measure the neural activity of insects is of significant interest to the field of insect toxicology and physiology since the nervous system is the target tissue of the majority of deployed insecticides¹. Yet, continued growth of fundamental and applied knowledge regarding the insect nervous system requires advanced neurophysiological techniques that are limited in feasibility, since current techniques are labor intensive and require a high expense, insect neural cell lines are limited, and/or there is limited access to the central synapses of most arthropods. Currently, characterization of most insect neural proteins requires the target to be cloned and heterologously expressed for subsequent drug discovery and electrophysiological recordings, as was described for insect inward rectifier potassium channels², insect ryanodine receptor³, mosquito voltage-sensitive K⁺ channels⁴, and others. To mitigate the requirement for heterologous expression and the potential for low functional expression, Bloomquist and colleagues aimed to induce a neuronal phenotype in cultured Spodoptera frugiperda (Sf21) cells as a novel method for insecticide discovery^5,6. These methods provide a valid approach for the development of new chemistry, yet they oftentimes create an insurmountable bottleneck for the characterization of pharmacological agents, identifying mechanisms of insecticide resistance, and characterization of fundamental physiological principles. Here, we describe an ex vivo method that enables the recording of electrical activity from a model insect that has malleable genetics^7,8,9 and known expression patterns of neural complexes^10,11,12 to enable the characterization of resistance mechanisms at the level of the nerve, the mode of action of newly developed drugs, and other toxicological studies.

The fruit fly, D. melanogaster, is a common model organism for defining insect neural systems or insecticide mechanism of action and has been established as a well-suited model organism for the study of toxicological¹³, pharmacological^14,15, neurophysiological¹⁶, and pathophysiological^17,18,19,20 processes of vertebrates. D.melanogaster is a holometabolous insect that performs complete metamorphosis, including a larval and pupal stage before reaching the reproductive adult stage. Throughout the developmental process, the nervous system undergoes significant remodeling at different life stages, but the larval CNS will be the focus of this methodology. The fully developed larval CNS is anatomically simple with thoracic and abdominal segments that are fused and form the ventral ganglion, which represents an array of repeated and almost identical neuromeric units^21,22. Descending motor nerves originate from the caudal end of the subesophageal ganglia and descend to innervate body wall muscles and visceral organs of the larvae. Figure 1 describes the gross anatomy of the larval Drosophila CNS.

The Drosophila blood-brain barrier (BBB) develops at the end of embryogenesis and is formed by subperineurial glial cells (SPG)²¹. The SPG cells form numerous filopodia-like processes that spread out to establish a contiguous, very flat, endothelial-like sheet that covers the entire Drosophila CNS²³. The Drosophila BBB has similarities to the vertebrate BBB, which includes preserving the homeostasis of the neural microenvironment by controlling the entry of nutrients and xenobiotics into the CNS²¹. This is a prerequisite for reliable neural transmission and function, yet the protection of the CNS by the BBB restricts the permeation of synthetic drugs, most peptides, and other xenobiotics^24,25, which introduces potential problems when characterizing potencies of small-molecule modulators. The method uses a simple transection to disrupt this barrier and provide ready pharmacological access to the central synapses.
The greatest strength of the described methodology is the simplicity, reproducibility, and relatively high-throughput capacity inherent to this system. The protocol is relatively easy to master, the setup requires little space, and only an initial financial input is necessary which is reduced to reagents and consumables. Further, the described method is completely amendable to record the central descending nerve activity of the house fly, Musca domestica²⁶.

Protokół

1. Equipment and Materials

1. Prepare the required components (listed in Table of Materials) of the electrophysiology rig to perform suction electrode recordings of the Drosophila CNS.
   NOTE: Prior to experimentation, it is necessary to construct chambers for dissection of the Drosophila CNS and to be used for bathing the ganglia in saline during recordings. A step-by-step outline of chamber construction is provided below.
2. Prepare the larval chamber.
   1. Melt the black wax using a hot plate.
   2. Pour 2 mL of melted wax into a chamber that has a maximum volume of 2 - 2.5 mL.
   3. Let the wax cool and harden for approximately 2 h.
   4. Use a razor blade to carve a hole in the hardened wax that will hold a volume of 500 µL, which has a surface area of approximately 0.5 cm² and a depth of approximately 0.25 cm.

2. Equipment and Software Configuration

NOTE: The setup of the extracellular recording is briefly described below.

1. Prepare equipment for dissection and recording.
   1. Position the tissue preparation, microscope, suction electrode, micromanipulators, light source inside the Faraday cage to reduce noise and eliminate extraneous electrical fields (Figure 2).
   2. Connect (preferably solder) ground and positive wires onto shielded alligator clips that will be connected to the bath and microelectrode holder, respectively.
      NOTE: Soldered and exposed wires can be covered with aluminum foil to reduce noise.
   3. Connect the ground and positive wires to input 1 of the AC/DC differential amplifier.
   4. Connect the data acquisition system to the AC/DC differential amplifier by connecting a Bayonet Neill-Concelman (BNC) cable from input 1 of the data acquisition system to the channel 1 output of the amplifier.
      NOTE: It is recommended to incorporate a 50/60 Hz noise eliminator between the data acquisition software and the amplifier. The use of a BNC T-connector is required.
   5. If desired, include an audio monitor into the setup by connecting input 1 of the audio monitor to input 1 of the data acquisition software.
   6. Fill the 10 mL syringe with saline and connect it to the pressure port of the electrode holder.
      NOTE: Ensure that no air bubbles exist between the syringe, the Ag/AgCl pellet, and the electrode opening.
2. Prepare the software for dissection and recording.
   NOTE: The outline of methods for software setup is based on the acquisition/analysis software listed in the Table of Materials which will digitize the raw electrical output and convert the data to spike frequency. However, other software can be used and multiple recording conversions can be used.
   1. Open the acquisition/analysis software.
   2. Click on "setup" from the main toolbar and select "channel settings," which will open a dialogue box.
   3. Reduce the number of total channels to three.
   4. For Channel 1, select a range of 100 mV. 
   5. For Channel 2, click on the "calculation" tab that will open a drop-down menu. Select "cyclic measurements," which will open a second dialogue box.
   6. Select channel 1 as the source. Then on the drop down menu measurement, select unit spike at events. Finally, in the detection settings area, from the drop down menu select simple threshold. 
   7. To convert the electrical activity into a rate plot expressed in hertz, the 3^rd channel must be set in arithmetic mode: On the input window, type in "1000*smoothsec(ch2,2)". Then select as the units drop down menu hertz.
      NOTE: Successful recordings can be performed with multiple different recording setups, but "Unit spike at events and simple threshold" is likely the easiest, since it allows for adjustment of the threshold above the background activity for each individual recording. Recordings under the setting of "Custom-Source Input" increase reproducibility of the data assuming the background activity is not different between recordings.
   8. Close dialogue boxes to return to the main screen. The y-axis should be expressed in Hz and the x-axis in time.

3. Dissect and Prepare the Larval Drosophila CNS

NOTE: Methods for larval CNS dissection are clearly illustrated in Hafer and Schedl²⁷, but these previously published methods reduce the length of the descending neurons that are important for measuring spike frequency. Here, an additional method is outlined to excise the larval CNS that maintains long, intact descending neurons.

1. Saline preparation.
   1. Prepare the dissection and recording saline to the following in mM²⁸: 157 NaCl, 3 KCl, 2 CaCl₂, 4 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES); titrate pH to 7.25.
2. Dissect the larval Drosophila CNS.
   1. Identify and extract a wandering third-instar Drosophila melanogaster from the culture vial and place into 200 µL of saline (Figure 3A).
   2. Grasp the mouth hooks with a pair of fine forceps and grasp the abdomen of the maggot with a second pair of forceps (Figure 3B).
      NOTE: Do not apply too much pressure to the abdomen as this can result in tearing of the thin cuticular layer of the maggot.
   3. Gently pull the mouth hooks and abdomen in different directions to separate the caudal end of the maggot from the head region and expose the viscera of the maggot (Figure 3C).
      NOTE: The CNS will be intertwined with the trachea and digestive tract. Do not cut the CNS away from any tissue to prevent cutting the descending peripheral nerves.
   4. Tease the CNS out of the digestive tract and trachea with forceps (Figure 3D).
   5. If necessary, disrupt the blood brain barrier by manually transecting the CNS posterior to the cerebral lobes with Vannas spring scissors.
      NOTE: This should be done based on the physiochemical properties of the chemical being used. The red line in Figure 4A is the suggested transection point and the transected CNS with intact descending peripheral nerve trunks shown in Figure 4B. The transected CNS is ready for experimentation after completion of step 3.2.5.

4. Extracellular Recording of Drosophila CNS.

1. Prepare the CNS for recording.
   1. Pull a glass pipette electrode from borosilicate glass capillaries to a resistance of 5-15 mΩ.
   2. Insert the transected CNS into the wax chamber that contains 200 µL of saline. Clamp an uncoated insect pin with the alligator clip soldered to the ground wire and insert the pin into the saline to complete the circuit.
   3. Using the micromanipulators, orient the electrode to the caudal end of the transected CNS. Eliminate recording of background noise by adjusting the threshold level in the acquisition/analysis software prior to contacting the peripheral nerve trunks.
   4. Draw any convenient peripheral nerves into the suction electrode by applying slight negative pressure on the syringe.
      NOTE: The baseline firing frequency is correlated to the number of neurons drawn into the electrode where more neurons oftentimes result in increased resistance of the electrode.
2. Begin extracellular recording of CNS descending nerve activity.
   1. Start the recording on the data acquisition software and monitor the baseline firing frequency. Allow the firing rate to equilibrate for 5 min prior to collecting baseline firing rate data.
   2. Discard the preparation and recording if the pattern of firing of control treatment is not a bursting pattern similar to that shown in Figure 5A.
      NOTE: Altered pattern of firing suggests abnormal or unstable activity of the central pattern generator, which can alter neural function.
   3. After 5 min, add 200 µL of saline + vehicle to bring the total volume of the chamber to 400 µL to begin recording control firing rates.
   4. After baseline has been established (3-5 min), withdraw 200 µL of saline and add 200 µL of the experimental agent solubilized in saline.
      NOTE: Dimethyl sulfoxide (DMSO) is the recommended solvent for lipophilic compounds and should not exceed 0.1% v/v.
   5. Apply drug concentrations in a serial manner (low to high concentrations) and record each concentration for a select period of time (determined by the investigator based on the chemical properties of the drug²⁹). Label this time point of drug application in the acquisition/analysis software by including a comment that includes the drug and the final concentration.
      NOTE: Firing activity of the CNS will decline by approximately 10-20% after 30-50 min after the dissection and therefore, appropriate untreated controls should be performed, and drug treatments should not exceed this time period.
3. Analyze the data.
   NOTE: Multiple analyses can be performed on the collected data, such as changes in action potential bursts²⁹, spike waveform amplitude and time course, and determining mean spike frequencies after drug treatment^26,30,31,32. The most common is determining the influence a drug has to the mean spike frequencies over a specified period of time, which is described below.
   1. Tabulate mean spike frequencies by selecting the entire region of interest (i.e., the drug treatment time period) and determine mean spike frequencies automatically through the "Datapad" located on the main toolbar of the acquisition/analysis software.
   2. Determine the mean spike frequency of the CNS after drug treatment to the mean spike frequency of the vehicle control (baseline). Calculate the percent change of firing rate after drug treatment by the formula: (Treated Frequency/Baseline Frequency) × 100.
   3. Use the mean spike frequencies or percent firing of control for each concentration to construct a concentration response curve with standard graphing software.
   4. Perform statistical analysis (e.g., unpaired t-test) to determine significance between time points, concentrations, or drug treatments.
      NOTE: There are two primary methods for generating and analyzing concentration-response curves. The first method is one concentration per individual preparation. Here, the spike rate of the single concentration is normalized to baseline spike rate for each preparation. The benefits are reduced run down of the preparation due to shortened recording time, while the pitfalls are increased dissection time because this method will consist of 5-7 individual CNS preparations per concentration, and larger error bars on the averaged data set. The second method is multiple concentrations per individual preparation. Here, the spike rate for each of the 3-5 concentrations are normalized to the same baseline spike rate. The benefits are less dissection time and less variability between replicates for drug concentrations, while the pitfalls are the requirement of a fast-acting drug and unknown impact of previous drug concentration treatments on subsequent chemical treatments.

Wyniki

Spontaneous activity of the descending peripheral nerves arising from the Drosophila central nervous system can be recorded using extracellular suction electrodes with consistent reproducibility. Spontaneous activity of the excised and transected Drosophila CNS produces a cyclical pattern of bursting with 1-2 s of firing with approximately 1 s of near quiescent activity. For example, the CNS is near quiescent (1-2 Hz) for 0.5-1 s, followed by a burst (100-400 Hz) for app...

Dyskusje

The details provided in the associated video and text have provided key steps in order to record the activity and spike discharge frequency of the Drosophila CNS ex vivo. The dissection efficacy is the most critical aspect of the method because short or few descending neurons will reduce the baseline firing rate that will result in large variances between replicates. However, once the dissection technique has been mastered, the data collected with this assay are highly reproducible and amendable for a w...

Materiały

Name	Company	Catalog Number	Comments
Drosophila melanogaster (strain OR)	Bloomington Drosophila Stock Center	2376
Vibration isolation table	Kinetic Systems	9200 series
Faraday Cage	Kinetic Systems	N/A
Dissecting Microscope on a Boom	Nikon	SMZ800N	Multiple scopes can be used; boom stand is critical
AC/DC differential amplifier	ADInstruments	AM3000H	The model 1700 can be used instead of the model 3000
audio monitor	ADInstruments	AM3300
Hum Bug Noise Eliminator	A-M Systems	726300
Data Acquisition System (PowerLab)	ADInstruments	PL3504	Multiple PowerLab models can be used.
Lab Chart Pro Software	ADInstruments	N/A - Online Download
Fiber Optic Lights	Edmund Optics	89-740	Different light sources can be used, but fiber optics are the most adaptable
Micromanipulator	World Precision Instruments	M325
Microelectrode Holder	World Precision Instruments	MEH715	Different models are acceptable
BNC cables	World Precision Instruments	multiple based on size
Glass Capillaries	World Precision Instruments	PG52151-4
Microelectrode Puller	Sutter Instruments	P-1000	Also can use Narashige PC-100
Black Wax	Carolina Biological Supply	974228
Non-coated insect pins, size #2	Bioquip	1208S2
Fince Forceps	Fine Science Tools	11254-20
Vannas Spring Scissors	Fine Science Tools	15000-03